Our overall goal is to determine the molecular mechanisms responsible for controlling the expression of genes for enzymes that are synthesized during the S phase of the cell cycle. In this study we will examine the expressiion of the gene for thymidylate synthetase (TS) in cultured mouse 3T6 fibroblasts experiencing serum induced transitions between the resting and growing states. We have recently shown that the cellular level of TS is nearly undetectable in resting 3T6 cells, but increases dramatically in serum stimulated cells during S phase. During the next few years, we will complete our analysis of the changes in TS activity in serum stimulated cells to define the conditions required to bring about the maximum changes in TS gene expression. We will isolate and characterize a fluorodeoxyuridine-resistant 3T6 cell line that overproduces TS (the target enzyme) and its mRNA by greater than 100-fold. We will determine if TS gene expression is regulated in the overproducing cell line in the same manner as in normal 3T6 cells. If so, the former will be used as a covenient model system for more detailed studies on the rate of synthesis of TS and its mRNA. TS will be purified from the overproducing cell line by affinity chromatography and characterized. Rabbit antibodies to the pure enzyme will be prepared and used to determine the in vivo and in vitro rates of synthesis of TS, and to purify TS mRNA by polysome immunoprecipitation. Recombinant DNA plasmids containing cDNA corresponding to TS mRNA will be isolated using standard recombinant DNA procedures. The cloned cDNA will be used in a variety of experiments to analyze the metabolism of DHFR mRNA and its precursors and to study the structure of and eventually isolate, the TS gene. We will also determine if TS overproduction is the result of gene amplification, and study the mechanism of gene amplification.